Objective To investigate the effect of retinoic acid on differentiation of periodontal ligament cells (PDLCs) and gingival fibroblasts(GFs). Methods The periodontal ligament cells and gingival fibroblasts were cultured, challenged with different concentrations of retinoic acid in medium and detected for the alkaline phosphatase (ALP) activity and its mRNA by biochemical technique, in situ hybridization and RT- PCR. Results ALP activity in normal PDLCs was higher than that in GFs. The mRNA signals were positive in PDLCs, and negative in GFs. After treated with different concentrations of retinoic acid, ALP activity of PDLCs was increased than that of the control, and its mRNA signals were enhanced, especially in 5×10- 6 mol/L. The treated GFs showed a slight increase of ALP activity and a weak band of mRNA signals only in 5×10- 6 mol/L concentration. Conclusion There were differences between PDLCs and GFs in differentiating into osteoblast- like cells.